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J. Biol. Chem., Vol. 279, Issue 28, 29050-29059, July 9, 2004

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Specificity of a Soluble UDP-Galactose:Fucoside 1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium^*

Catherine Ketcham, Fei Wang, Suzanne Z. Fisher, Altan Ercan¶, Hanke van der Wel¶, Robert D. Locke||, k. Sirajud-Doulah||, Khushi L. Matta||, and Christopher M. West¶**

From the Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235, the ^||Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263, and the ^¶Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Skp1 is an adaptor-like protein in E3^SCF-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gal1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn²⁺ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gal1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to 1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K_m and 35-fold higher V_max compared with a simple -fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V_max advantage, but still much less active than Skp1 itself because of a K_m difference. These findings indicate that -GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gal1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Received for publication, December 18, 2003 , and in revised form, April 20, 2004.

^* This work was supported in part by National Institutes of Health Grant R01-GM37539. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK 73104. Tel.: 405-271-2227 (ext. 1247); Fax: 405-271-3139; E-mail: Christopher-West{at}ouhsc.edu.

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